ABSTRACT The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of theEscherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (α-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue α-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to α-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes toSalmonella resistance to innate immunity.

A remote flow cell based on a single strand of fused-silica fiber optic was built for UV absorbance detection with a packed capillary HPLC system, using commercially available pumps, detection electronics (Shimadzu) and fittings. This 'off-column' flow cell design is applicable to both pressure and electro-osmotically driven systems. The goals were to minimize the linearity and light leakage problems that often limit the performance of UV absorbance detection with capillary chromatography. A linear dynamic range of 10(3) (reserpine, lambda = 220 nm), and a concentration detection limit of 5.1 x 10(-8) mol l(-1) were observed. Baseline noise was measured at 3.5 x 10(-5) absorbance units (AU), with a standard deviation of 1.7 x 10(-5) AU. The illuminated volume of approximately 3 nl was optimized for capillaries with inner diameters in the range 50-100 mu m, and flow rates from 100 nl min(-1) to 1 mu l min(-1). These modifications of readily available instrumentation have allowed the construction of a practical system for fractionating complex mixtures of peptides in small amounts, prior to mass spectrometry or additional wet chemistry steps.

An interface was developed for mass spectrometry based on de induction. Ions were produced as liquid flowed through a concentric cylindrical capacitor at room temperature. No externally applied high voltages affecting the spray tip were used, thus eliminating corona discharge problems in negative ion mode. The voltages applied to the capacitor were lower than those used for electrospray on the same instrument. Stable signals were observed for positive ions infused at flow rates from 50 nL/min to 200 mu L/min, anions in the range 50-500 nL/min, without addition of sheath liquid or gas. The minimum concentration required to generate an interpretable product ion spectrum from the [M - 2H](2-) precursor, for infused Salmonella typhimurium diphosphorylated lipid A, was improved by a factor of similar to 500. Positive ion capillary LC/MS results for a horse apomyoglobin tryptic digest were similar to those observed for an optimized electrospray interface. Factor XIII b subunit, an 83-kDa protein, yielded an interpretable positive ion multiple charge envelope, under buffer conditions that did not allow collection of useful data with our low-now electrospray source. Mass spectra were also generated for a single-stranded DNA 15-mer and 2', 4'-dideuteriotrifluoropropranolol, a small molecule that had been difficult to ionize by either electrospray or APCI.

Like many other lepidoptera, Manduca sexta synthesizes juvenile hormones (JH) that differ from classic isoprenoids by having one or more ethyl, instead of methyl, branches attached to the hydrocarbon chain. This difference originates from the utilization of homomevalonate in the place of mevalonate for synthesis of the different forms of JH. No other ethyl-branched homoisoprenoid has been found in lepidopteran insects. We reported earlier that only methyl-branched dolichols were detected in M. sexta larvae. We have now isolated ubiquinones from the embryos of M. sexta and then analyzed them by chemical ionization mass spectrometry. No ubiquinone homologue with an ethyl-branched side chain was detected. The predominant homologue found is ubiquinone Q-9 entirely with methyl-branches, with only a trace amount of ubiquinone Q-10. These results suggest that JH are synthesized by specialized enzymatic mechanisms in the corpora allata, while other major isoprenoids are synthesized by the more common enzymes present in eukaryotic systems.

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor-urotensin-sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of approximately 1 microM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29-R30 and R30-A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

Electrospray ionization sources, used with triple quadrupole mass spectrometers from PE/Sciex (API III+), Micromass (Quattro II), and Finnigan (TSQ 7000), were modified with a 35-gauge stainless steel needle. The dimensions of the needle were 63 microm i.d. by 145 microm o.d. with variable length, depending on the specific instrument. This modification led to enhanced signal stability, improved signal/noise ratios, and lowered sample consumption for a wide range of peptides. Stable baselines were observed with flow rates in the range of 50 nL/min to 5 microL/min. An alternative design, based on a metal wire housed within a fused silica capillary, led to the most stable signals of all during infusion, but caused excessive peak broadening with capillary chromatography. The Finnigan interface was further modified with an external postcolumn addition tee, used in conjunction with capillary liquid chromatography columns of 30 and 50 microm internal diameter. The best results with the modified Finnigan interface were acquired using the 50-microm column at a flow rate of 150 to 200 nL/min.

We isolated and analyzed dolichols from fifth stadium larvae of Manduca sexta, in an effort to determine if M. sexta produces ethyl-branched dolichols. Like other lepidopterans, this animal is known to produce the ethyl-branched, homoisoprenoid juvenile hormones. However, we were unable to detect any ethyl-branched dolichol homologues. Manduca sexta larvae at this stage do contain the common eukaryotic dolichols, principally dolichols 17, 18, 19 and 20 in an ∼ 3:7:5:2 ratio. Analysis of the yeast-containing artificial diet showed the presence of dolichols 13, 14, 15, 16, and 17 in an ∼ 2:6:8:6:3 ratio. We attempted to determine whether M. sexta synthesized dolichols de novo; preliminary experiments using [5-3Hlmevalonate incubated with two M. sexta tissues in vitro were unsuccessful. In light of earlier studies on dolichol identity in two fly species, we conclude that insects can synthesize dolichols, even though they are known to be incapable to synthesizing sterols from acetate.

A remote flow cell has been interfaced to a scanning uv-visible absorbance detector for the detection of gas chromatography (GC) analytes. The unique optical bench, detection scheme, and thermal control allow low-noise operation at flow cell temperatures of 350 °C. Noise of 1×10−5 a.u. (peak to peak over a 1-min period) and drift at the 1×10−4 a.u./h level facilitate detection of coumarin in the gas phase at 40 pg/s. Flow cell volume was optimized for use with capillary GC columns. An effective band pass of <3 nm provides spectral resolution which shows solute absorbance fine structure to aid in compound identification.